The molecular structures of proteins involved in two aspects of DNA replication and repair, DNA unwinding and DNA polymerization, will be studied utilizing the techniques of x-ray crystallography. DNA bound and free forms of the proteins, as well as site-directed mutants, will be investigated. During the last grant period, four protein systems involved in these processes were studied: the DNA polymerase I from Thermus aquaticus, Escherichia coli single strand DNA-binding protein SSB, and the E.coli dimeric DNA-helicases Rep and UvrD. These proteins will be studied further, while the structural studies of one additional protein system, the DNA polymerase delta holoenzyme from Saccharomyces cerevisiae, will be initiated. The Taq DNA polymerase is involved in DNA repair, while the Polymerase delta holenzyme is the major replicative enzyme in eukaryotes. The Taq DNA polymerase is also an important biotechnological tool, used extensively in DNA sequencing and in the polymerase chain reaction. The crystal structures of nine different ligated states of an active N-terminal deletion of Taq polymerase corresponding to the Klenow fragment of E. coli of DNA polymerization have been determined. Additional complexes will be studied, as well as the full-length Taq polymerase which contains at its N-terminus a 5'-3' exonuclease activity. Studies of components of the polymerase delta holoenzyme will be initiated, which will shed light into the process of replicative DNA polymerization, which has been conserved from phage T4 to human. DNA unwinding is a fundamental process in DNA replication and repair. Several human diseases, including xeroderma pigmentosum and Cockayne's syndrome, involve defects in proteins that possess helicase activity. E. coli Rep and UvrD are involved in active unwinding of DNA, while E. coli SSB is involved in passive DNA helix destabilization. Rep and UvrD both function as dimmers and are motor proteins which use the energy derived from ATP-binding and hydrolysis to cycle kinetically through conformational states. The structures of the Rep helicase bound to a single-stranded DNA, as well as the structure of the DNA binding domain of E. coli SSB were determined. Structures of UvrD, as well as of complexes of SSB with DNA, are in the process of being solved. Various functionally-relevant complexes of these proteins, kinetically trapped at various stages of unwinding, will be studied. Such structures will provide information on the mechanism of active unwinding by DNA-helicases and of passive unwinding by helix-destabilizing SSB proteins.